Mechanism of piRNA-mediated recognition and silencing of foreign genes

piRNA介导的外源基因识别和沉默机制

基本信息

批准号：
9197347
负责人：
Heng-Chi Lee
金额：
$ 24.9万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9197347
关键词：
Affinity Algorithms Animal Testing Animals Applied Genetics Base Pairing Binding Biochemical Biochemical Genetics Biogenesis Bioinformatics Biological Assay Biological Process Caenorhabditis elegans Complex DNA Transposable Elements Data Defect Defense Mechanisms Development Disease Epigenetic Process Exhibits Failure Family Fellowship Fertility Gene Expression Gene Silencing Genes Genetic Genetic Determinism Genetic Screening Genome Genome Stability Genomic Instability Goals Health Heterochromatin In Vitro Infertility Invaded Lead Malignant Neoplasms Maps Measures Mediating Mentors MicroRNAs Modeling Molecular Genetics Names Nematoda Nucleic Acids Nucleotides Organism Pathogenesis Phase Play Protein Array Protein Family Proteins RNA RNA Interference RNA Sequences RNA biosynthesis RNA-Directed RNA Polymerase Reagent Recruitment Activity Reporter Research Role Scanning Seeds Site Small RNA System Testing Transgenes Transgenic Animals Untranslated RNA Virus base epigenetic regulation forward genetics genetic approach genome sequencing in vitro testing in vivo insight member mutant novel piRNA programs skills tool transcriptome whole genome

项目摘要

DESCRIPTION (provided by applicant): Invasive foreign nucleic acids such as transposons and viruses pose a threat to all organisms, potentially causing genome instability and disease, including cancer and infertility. Fortunately, organisms have evolved a vast array of protein and nucleic acid-based mechanisms to defend against intruders. In animals, germline-expressed small RNAs known as piRNAs interact with PIWI-clade Argonaute proteins, which together promote genome stability and fertility, in part by silencing transposable elements. Yet piRNAs exhibit remarkable sequence diversity and often map to unique sequences in the genome. In the nematode worm C. elegans, for example, tens of thousands of piRNAs are individually expressed from mini-genes and lack perfectly complementary targets elsewhere in the genome. Therefore the function of piRNAs has remained mysterious. We have recently shown that piRNAs can recognize their targets via imperfect base pair interactions and trigger stable and heritable epigenetic silencing, which can be maintained by RNAi and heterochromatin factors, independently of piRNAs. The goal of this proposal is to answer several fundamental questions regarding this RNA-mediated defense system, including: How do piRNAs recognize foreign nucleic acids? What are the key molecular and genetic determinants of piRNA targeting? How does piRNAs targeting trigger an epigenetic silencing? During the K99 (mentored) phase of this fellowship, I will develop biochemical assays and genetic reporters to understand how piRNAs recognize their targets. This information will aid the development of bioinformatics tools to predict and analyze in vivo piRNA targets. In addition, I will perform a genetic screen to identify factors required for piRNA- mediated targeting and silencing. During my R00 phase, I will further use a combination of biochemical, genetic and bioinformatics approaches to study the biogenesis of piRNAs and mechanism of piRNA-induced gene silencing. The studies proposed here will provide important insights into role of piRNAs in genome surveillance and epigenetic regulation and their role in disease pathogenesis. Furthermore, I will obtain reagents, tools and skills necessary to launch my independent research program.

描述（由申请人提供）：侵入性外来核酸（例如转座子和病毒）对所有生物体构成威胁，可能导致基因组不稳定和疾病，包括癌症和不育症。幸运的是，生物体已经进化出大量基于蛋白质和核酸的机制来防御入侵者。在动物中，种系表达的小 RNA（称为 piRNA）与 PIWI 进化枝 Argonaute 蛋白相互作用，共同促进基因组稳定性和生育力，部分是通过沉默转座元件。然而，piRNA 表现出显着的序列多样性，并且通常映射到基因组中的独特序列。例如，在线虫秀丽隐杆线虫中，数以万计的 piRNA 由小基因单独表达，并且在基因组其他地方缺乏完美互补的靶标。因此piRNA的功能仍然是个谜。我们最近表明，piRNA 可以通过不完美的碱基对相互作用识别其靶标，并触发稳定且可遗传的表观遗传沉默，这种沉默可以通过 RNAi 和异染色质因子来维持，独立于 piRNA。该提案的目标是回答有关 RNA 介导的防御系统的几个基本问题，包括：piRNA 如何识别外来核酸？ piRNA 靶向的关键分子和遗传决定因素是什么？ piRNA 靶向如何触发表观遗传沉默？在该奖学金的 K99（指导）阶段，我将开发生化检测和基因报告基因，以了解 piRNA 如何识别其目标。这些信息将有助于开发生物信息学工具来预测和分析体内 piRNA 靶标。此外，我将进行基因筛查来识别 piRNA 介导的靶向和沉默所需的因素。在R00阶段，我将进一步结合生化、遗传学和生物信息学方法来研究piRNA的生物发生以及piRNA诱导的基因沉默的机制。这里提出的研究将为 piRNA 在基因组监测和表观遗传调控中的作用及其在疾病发病机制中的作用提供重要见解。此外，我将获得启动我的独立研究计划所需的试剂、工具和技能。